Calcineurin, also known as protein phosphatase 2B, was first identified in the bovine brain. It represents a small family of calcium and calmodulin dependent serine/threonine protein phosphatases. It is expressed in all mammalian tissues examined, and is most abundant in the brain. In lymphocytes, calcineurin is the major soluble calmodulin-binding protein. Calcineurin is a heterodimer consisting of a catalytic subunit (A; 61 kD) and a regulatory subunit (B; 19 kD). The A subunit contains a catalytic domain, a carboxyl-terminal inhibitory domain, a B subunit binding site, and a camodulin binding site. The phosphatase activity of the A subunit is regulated by CA2+ through both calmodulin and the B subunit. The B subunit has only a Ca2+ dependent regulatory activity and does not have any phosphatase activity. There are two genes encoding closely related (about 80% identical) A subunit isoforms, Axcex1 and Axcex2, in the mouse, human, and rat genomes. The xcex1 isoform is the predominant isoform found in brain, thymus, and T cells. The Axcex1 and Axcex2 isoforms have distinct cellular distribution in the brain, with Axcex1 most abundant in the hippocampus, cerebral cortex, cerebellum, and striatum. The differential distributions of the two isozymes suggest they may each have specific functions in modulating neuronal activities. The physiologic functions of the different calcineurin A isoforms are not yet defined.
The present invention relates to a method of identifying drugs or agents which have immuno-suppressive effects through or as a result of their effect on calcineurin, including drugs which affect the calcineurin Axcex1 (CNAxcex1) subunit or the calcineurin Axcex2 (CNAxcex2) subunit. It particularly relates to methods of identifying drugs which inhibit the phosphatase activity of calcineurin. The present invention further relates to a method of identifying drugs which overcome, prevent or reduce (partially or totally) the neurotoxic or other adverse effects of immuno-suppressant drugs, such as cyclosporin A (CsA) and FK506, which exert their effects by inhibiting calcineurin phosphatase activity.
In addition, the present invention relates to a method of identifying drugs which reduce (partially or totally) phosphorylation of the microtubule-associated protein tau, in the nervous system of a mammal; a method of identifying drugs which reduce (partially or totally) paired helical filament formation in the nervous system of a mammal; and a method of identifying drugs which reduce (partially or totally) formation of paired helical filaments, amyloid deposits or both. Such drugs are useful in reducing the extent to which Alzheimer""s disease occurs, reducing the rate at which Alzheimer""s disease progresses or preventing its occurrence.
The present invention also relates to transgenic non-human mammals, such as rodents and particularly mice, which lack a functional calcineurin gene and, thus, have disrupted calcineurin expression. In one embodiment, transgenic non-human mammals of the present invention lack a functional calcineurin Axcex1 (CNAxcex1) subunit gene, a functional calcineurin Axcex2 (CNAxcex2) subunit gene or both CNAxcex1 and CNAxcex2 subunit genes. In a further embodiment, transgenic non-human mammals (e.g., rodents such as mice and rats) lack a functional calcineurin gene (e.g., calcineurin subunit Axcex1 gene, calcineurin subunit Axcex2 gene) and express human tau protein. In such transgenic mammals, hyperphosphorylation of human tau protein is expressed and polymerizes, resulting in formation of paired helical filaments that make up neurofibrillary tangles in the brain. A third type of transgenic non-human mammal (e.g., rodents, such as mice and rats) lacks a functional calcineurin gene, expresses human tau protein and overexpresses human amyloid precursor protein and human Alzheimer Axcex2 protein. Such transgenic mammals exhibit both of the pathological lesions of Alzheimer""s diseasexe2x80x94amyloid deposits and paired helical filaments (which make up the neurofibrillary tangles that accumulate in brain neurons in Alzheimer""s disease)xe2x80x94and serve as an improved model for Alzheimer""s disease in which to identify drugs or agents which will reduce (partially or totally) the pathological lesions.
As described herein, a transgenic non-human mammal which lacks a functional calcineurin (CN) gene produces greatly increased amounts of hyperphosphorylated tau protein. The transgenic non-human mammal of the present invention can be used to identify drugs or agents which have immuno-suppressive effects through or as a result of their effect on CN, including drugs or agents which affect the calcineurin Axcex1 (CNAxcex1) subunit or the calcineurin Axcex2 (CNAxcex2) subunit. In addition, further transgenic mammals of the present invention, described herein, can be used to identify agents which are useful in reducing phosphorylation of tau protein and production of pathological lesions characteristic of Alzheimer""s Disease.
In one embodiment, the present invention relates to a method of identifying an agent that reduces the phosphorylation of tau protein in the nervous system of a mammal, comprising the steps of a) administering to a transgenic non-human mammal which lacks a functional CN gene, an agent to be assessed for its ability to reduce phosphorylation of tau protein; b) determining the extent to which phosphorylation of tau protein occurs in the nervous system of the transgenic non-human mammal to which the agent is administered; and c) comparing the extent determined in b) to the extent to which phosphorylation occurs in the nervous system of an appropriate control. If phosphorylation occurs to a lesser extent in the nervous system of the transgenic non-human mammal to which the agent is administered than in the nervous system of the control, the agent reduces phosphorylation of tau protein.
In another embodiment, the present invention relates to a method of identifying an agent which reduces paired helical filament (PHF) formation in the nervous system of a mammal, comprising the steps of: a) administering to a transgenic non-human mammal which lacks a functional CN gene and expresses human tau protein, an agent to be assessed for its ability to reduce PHF formation; b) determining the extent to which PHF formation occurs in the nervous system of the transgenic non-human mammal to which the agent is administered; and c) comparing the extent determined in b) to the extent to which PHF formation occurs in the nervous system of an appropriate control, wherein if PHF formation occurs to a lesser extent in the nervous system of the transgenic non-human mammal to which the agent is administered than in the nervous system of the control, the agent reduces PHF formation. In another embodiment, the present invention relates to a method of identifying an agent which reduces a lesion characteristic of Alzheimer""s disease in the nervous system of a mammal comprising the steps of: a) administering to a transgenic non-human mammal which lacks a functional CN gene, expresses a human tau protein, and overexpresses the human amyloid precursor protein and the human Alzheimer Axcex2 protein, an agent to be assessed for its ability to reduce a lesion characteristic of Alzheimer""s disease; b) determining the extent to which the lesion occurs in the nervous system of the transgenic non-human mammal to which the agent is administered; and c) comparing the extent determined in b) to the extent to which the lesion occurs in the nervous system of an appropriate control; wherein if the lesion occurs to a lesser extent in the nervous system of the transgenic non-human mammal to which the agent is administered than in the nervous system of the control, the agent reduces a lesion characteristic of Alzheimer""s disease.
The pathological lesions characteristic of Alzheimer""s Disease which can be reduced in a mammal using agents identified by the method of the present invention include paired helical filament (PHF) formation and amyloid deposits. In addition, the phosphorylation of tau protein associated with Alzheimer""s disease can be reduced using agents identified by the methods of the present invention.
The present invention further relates to a method of identifying an agent that reduces the phosphatase activity of calcineurin AP subunit gene in the nervous system of a mammal, comprising the steps of: a) administering to a transgenic non-human mammal which lacks a functional calcineurin Axcex2 subunit gene, an agent to be assessed for its ability to reduce the phosphatase activity of calcineurin Axcex2 subunit; b) determining the calcineurin Axcex2 subunit phosphatase activity present in cells in the nervous system of the transgenic non-human mammal to which the agent is administered; and c) comparing the calcineurin Axcex2 phosphatase activity determined in b) to the calcineurin Axcex2 phosphatase activity in cells in the nervous system of an appropriate control, wherein if calcineurin Axcex2 phosphatase activity is present to a lesser extent in the nervous system of the transgenic non-human mammal to which the agent is administered than in the nervous system of the control, the agent reduces phosphatase activity of calcineurin Axcex2 subunit.
The transgenic non-human mammal which lacks a functional CN gene includes mammals in which the CN gene is not present in the genome and mammals in which the structural or functional activity of the CN gene present in the genome of the mammal has been disrupted (both types are referred to as calcineurin knockout mammals). The CNAxcex1 subunit gene and/or the CNAxcex2 subunit gene can be removed or functionally disrupted for use in the present invention. For example, as described in Example 1, the genome of a non-human mammal can be recombined with a sequence which becomes inserted into the exon encoding the CNAxcex1 gene of the animal, resulting in disruption of CNAxcex1 expression. Other methods of producing a CN knockout mammal for use in the present invention can be determined by one of skill in the art using routine experimentation.
A suitable mammal for use in the present invention is a mammal, which upon removal of the CN gene or disruption of the function of the CN gene, produces increased amounts of hyperphosphorylated tau protein. Transgenic non-human mammals of the present invention include rodents, (e.g., rats, mice) and primates.
In the method of the present invention, determination of the ability of an agent to reduce the lesions associated with Alzheimer""s Disease is detected in the nervous system of the transgenic mammal. In particular, the effect of the agent to be assessed can be determined in the central nervous system of the transgenic mammal. For example, the effect of the agent to be assessed can be determined in the brain of the transgenic mammal.
The methods used to determine the ability of an agent or drug to reduce a lesion characteristic of Alzheimer""s disease, which includes phosphorylation of tau protein, are routine methods known to those of skill in the art. For example, as described in Example 1, determination of the extent to which phosphorylation of the tau protein occurs in the transgenic non-human mammal of the present invention can be determined using anti-PHF antibodies. Anti-PHF antibodies can also be used to determine the extent to which PHF formation occurs. Assessing reduction of amyloid deposits can be determined using anti-xcex2 protein, thioflavin S or Congo Red. In addition, behavioral observations of the transgenic mammal to which an agent has been administered can be used to determine the ability of the agent to reduce lesions characteristic of Alzheimer""s disease, including phosphorylation of tau protein.
A suitable control is a transgenic non-human mammal which has the same characteristics as the transgenic animal to which an agent being assessed is administered (i.e., the test transgenic non-human mammal). The test and control non-human mammals are maintained under the same conditions; they differ only in the presence (test animal) or absence (control animal) of the agent being assessed. For example, a suitable control used to compare the results achieved with the agent or drug to be assessed, is a transgenic non-human mammal which lacks a functional CN gene, a transgenic non-human mammal which lacks a functional CN gene and expresses a human tau protein or a transgenic non-human mammal which lacks a functional CN gene, expresses a human tau protein and overexpresses the human APP and the human Alzheimer Axcex2 proteins in the absence of the agent being assessed. For example, in the embodiment for identifying an agent that reduces the phosphorylation of tau protein in the nervous system of a mammal, a suitable control is a transgenic non-human mammal which lacks a functional CN gene. The amount of phosphorylation of tau protein in the control transgenic non-human mammal is determined in the absence of the agent being assessed. Other appropriate controls can be a corresponding wildtype mammal or other control determined by those of skill in the art using no more than routine experimentation.
The present invention further relates to a transgenic non-human mammal which lacks a functional calcineurin gene. In addition, the present invention relates to a transgenic non-human mammal which lacks a functional calcineurin gene and expresses the human tau protein. Further, the present invention relates to a transgenic non-human mammal which lacks a functional calcineurin gene, expresses the human tau protein and overexpresses the human amyloid precursor protein (APP) and the human Alzheimer Axcex2 protein.
Pathological lesions that characterize the Alzheimer""s disease brain and cause the neurodegeneration that leads to dementia include the extracellular amyloid deposits and the intracellular neurofibrillary tangles which are composed of bundles of paired helical filaments as seen in the electron microscope. The amyloid deposits are composed primarily of an approximately 42 amino acid peptide termed Axcex2 that is derived from the larger precursor protein termed amyloid precursor protein, or APP. The paired helical filaments are composed primarily of the microtubule-associated protein tau that has become abnormally modified with extra phosphate groups (hyperphosphorylated). The relative contribution of these two lesions to the neuronal cell death is not yet known, but both are believed to be important. The correlation with neuronal cell death and dementia is highest with the appearance of the neurofibrillary tangles containing paired helical filaments in neurons. It is, therefore, accepted as axiomatic in the field that a successful treatment for Alzheimer""s disease will need to prevent or reduce the formation of paired helical filaments or remove filaments already formed.
At the present time, no rodent model exists that generates paired helical filaments or accumulates hyperphosphorylated tau protein. The Exemplar/Athena transgenic mouse model for Alzheimer""s disease overexpresses a mutant form of the APP gene (associated with familial Alzheimer""s disease) and shows some synaptic loss and the accumulation of amyloid, but it does not produce paired helical filaments and does not show clear learning disorders (Games, D., et al., Nature, 373:523-527 (1995). It is therefore of importance to develop a mouse model in which paired helical filaments can form. Such a model would serve as a target for testing potential Alzheimer""s therapeutic agents designed to reduce or prevent the formation of paired helical filaments. In addition, an animal model, preferably rodent, that shows both amyloid deposits and paired helical filaments would most closely resemble human Alzheimer""s disease and would allow the testing of therapeutic agents directed at reducing both of the pathological legions of Alzheimer""s disease.
On the basis of the discovery that the calcineurin knockout mouse produces greatly increased amounts of hyperphosphorylated tau protein, the mouse model for Alzheimer""s disease in which a hyperphosphorylated form of the human tau protein is expressed, hyperphosphorylated human tau protein and the accumulation of paired helical filaments is exhibited and the Alzheimer amyloid deposits of xcex2 protein and the paired helical filaments of hyperphosphorylated tau protein is expressed is generated as described below.
The calcineurin knockout mice is mated to a mouse line homozygous for the expression of a human tau protein. These latter mice have been generated by standard transgenic technology in which the human tau protein is injected into fertilized mouse oocytes in a construct that allows its expression under the control of the human thy 1 promoter. In these animals, the transgenic human tau protein is present in nerve cell bodies, axons, and dendrites and is partially hyperphosphorylated at the appropriate sites for producing paired helical filaments, but not to the degree in the mouse tau protein in the calcineurin knockout mouse line. Mating of these two animals will generate progeny, all of whom will carry a knocked out calcineurin gene on one chromosome, a normal calcineurin gene on the homologous chromosome, and half of whom will carry the human tau transgene.
The genotype of the progeny is determined by removing a small section of the tail, preparing DNA, and carrying out either a Southern Blot or PCR analysis to determine that they all carry one knocked out calcineurin gene and that 50% carry the human tau transgene. The progeny carrying the human tau sequence are grown to adulthood and inter-mated to generate a new set of progeny, 25% of whom are, by Mendelian laws, homozygous for the knocked out calcineurin gene and either homozygous or heterozygous for the human tau transgene. The genotypes of these animals is determined as before by analysis of tail DNA. Animals carrying the knocked out calcineurin gene in homozygous state plus the human tau transgene in either the heterozygous or homozygous state are further analyzed. Animals whose genotypes have been confirmed by the analysis of tail DNA are allowed to reach maturity and inter-mated to generate a line of animals that continues to have the correct genotypes. Mice at different ages are perfused with fixative and subjected to immunocytochemistry and electron microscopy to confirm that they express human tau protein and do not express the calcineurin protein.
Phosphorylation-sensitive antibodies are used as described for the calcineurin knockout mouse to confirm that the human tau protein is hyperphosphorylated due to the lack of calcineurin in its environment. Particular focus is placed on the hippocampus which has previously been shown to be an area of high calcineurin expression and the largest increase in hyperphosphorylation of tau due to the calcineurin knockout mutation. Neurofibrillary tangles are identified by modified Bielchowsky silver stain and by anti-PHF antibodies and are confirmed by electron microscopic identification. The protein expression studies are complemented by northern blot analysis to confirm that the calcineurin gene in these animals is not expressed.
Once a mouse line has been generated, it expresses hyperphosphorylated human tau protein and preferably paired helical filaments in the neurons of the hippocampus. The mice can be used in several ways. First, they can be used directly to screen for therapeutic agents that reduce the hyperphosphorylation of tau and the production of paired helical filaments. They can also be used to test putative therapeutic agents for their efficacy in preventing the hyperphosphorylation of tau and the formation of paired helical filaments. these mice can also be used to determine the ideal dose of a putative therapeutic agent for Alzheimer""s disease.
The mice can also be used to generate a further improved animal model for Alzheimer""s disease. For this use, homozygous mice which lack a functional calcineurin gene (mice homozygous for the knocked out calcineurin gene) and which express the human tau gene so that hyperphosphorylated human tau protein, and preferably PHF, is produced in the brain are mated to the Exemplar/Athena APP transgenic mouse that overexpresses the APP protein and the Alzheimer Axcex2 protein and, as a consequence, produces amyloid deposits. The purpose of this cross is to generate progeny that have all of the characteristics of Alzheimer""s disease, namely hyperphosphorylated tau, paired helical filaments, and amyloid deposits. The progeny of this cross are analyzed as before using tail DNA to confirm their genotype. For example, two heterozygous animals are crossed, one expressing the human tau transgene and one expressing a human APP transgene. Tail DNA analysis is carried out to determine which of the progeny carry both transgenes. If, on the other hand, the mating is between a homozygous version of the APP transgenic mouse and a homozygous version of the human tau transgenic mouse (of course already combined with the homozygous calcineurin knocked mutation), then technically the tail DNA analysis should not be necessary but is carried out nonetheless in case the germ line of any of the mice has lot any of the transgenes. The progeny of this cross thus carry two human transgenes, one for APP and one for tau under different promoters but both expressed in the nervous system, plus a homozygous knockout mutation in the calcineurin gene.
These mice will produce amyloid deposits and hyperphosphorylated tau/paired helical filaments, thus satisfying the two major criteria for an Alzhemer""s animal model. The mice can be used to screen for the test putative therapeutic agents for Alzheimer""s disease. They can also, as will the mice mentioned above, be used to test putative diagnostic tests for Alzheimer""s disease including, but not limited to, analysis of the tropicamide hypersensitivity of the pupil, the presence of key protein such as antichymotrypsin, APP, Axcex2, and hyperphosphorylated tau in the serum and/or cerebrospinal fluid.